Jean Sévigny
Université Laval, Microbiology and Immunology, Faculty Member
Research Interests: Immunology, Membrane Proteins, Macrophages, Apoptosis, Medicine, and 15 moreCell line, Platelet aggregation, Carbon Monoxide, Mice, Animals, Male, Heme Oxygenase, Rats, Monocytes, Experimental Medicine, Graft Rejection, Heart Transplantation, Environmental Exposure, Acute Disease, and Medical and Health Sciences
ATP is both an important mediator of physiological gut functions such as motility and epithelial function, and a key danger signal that mediates cell death and tissue damage. The actions of extracellular ATP are regulated through the... more
ATP is both an important mediator of physiological gut functions such as motility and epithelial function, and a key danger signal that mediates cell death and tissue damage. The actions of extracellular ATP are regulated through the catalytic functions extracellular nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3, and -8, which ultimately generate nucleosides. Ectonucleotidases have distinct cellular associations, but the specific locations and functional roles of individual NTPDases in the intestine are still poorly understood. Here, we tested the hypothesis that differential and cell-selective regulation of purine hydrolysis by NTPDase1 and -2 plays important roles in gut physiology and disease. We studied Entpd1 and Entpd2 null mice in health and following colitis driven by 2% dextran sulfate sodium (DSS) administration using functional readouts of gut motility, epithelial barrier function, and neuromuscular communication. NTPDase1 is expressed by immune cells, and the ablation of Entpd1 altered glial numbers in the myenteric plexus. NTPDase2 is expressed by enteric glia, and the ablation of Entpd2 altered myenteric neuron numbers. Mice lacking either NTPDase1 or -2 exhibited decreased inhibitory neuromuscular transmission and altered components of inhibitory junction potentials. Ablation of Entpd2 increased gut permeability following inflammation. In conclusion, the location- and context-dependent extracellular nucleotide phosphohydrolysis by NTPDase1 and -2 substantially impacts gut function in health and disease. NEW & NOTEWORTHY Purines are important mediators of gastrointestinal physiology and pathophysiology. Nucleoside triphosphate diphosphohydrolases (NTPDases) regulate extracellular purines, but the roles of specific NTPDases in gut functions are poorly understood. Here, we used Entpd1- and Entpd2-deficient mice to show that the differential and cell-selective regulation of purine hydrolysis by NTPDase1 and -2 plays important roles in barrier function, gut motility, and neuromuscular communication in health and disease.
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Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) are cell surface-located transmembrane ecto-enzymes of the CD39 superfamily which regulate inflammation and tissue repair by catalyzing the phosphohydrolysis of extracellular... more
Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) are cell surface-located transmembrane ecto-enzymes of the CD39 superfamily which regulate inflammation and tissue repair by catalyzing the phosphohydrolysis of extracellular nucleotides and modulating purinergic signaling. In the liver, NTPDase2 is reportedly expressed on portal fibroblasts, but its functional role in regulating tissue regeneration and fibrosis is incompletely understood. Here, we studied the role of NTPDase2 in several models of liver injury using global knockout mice. Liver regeneration and severity of fibrosis were analyzed at different time points after exposure to carbon tetrachloride (CCl4) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or partial hepatectomy in C57BL/6 wild-type and globally NTPDase2-deficient (Entpd2 null) mice. After chronic CCl4 intoxication, Entpd2 null mice exhibit significantly more severe liver fibrosis, as assessed by collagen content and histology. In contrast, deleti...
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Heme oxygenase 1 (HO-1) inhibits apoptosis by regulating cellular prooxidant iron. We now show that there is an additional mechanism by which HO-1 inhibits apoptosis, namely by generating the gaseous molecule carbon monoxide (CO).... more
Heme oxygenase 1 (HO-1) inhibits apoptosis by regulating cellular prooxidant iron. We now show that there is an additional mechanism by which HO-1 inhibits apoptosis, namely by generating the gaseous molecule carbon monoxide (CO). Overexpression of HO-1, or induction of HO-1 expression by heme, protects endothelial cells (ECs) from apoptosis. When HO-1 enzymatic activity is blocked by tin protoporphyrin (SnPPIX) or the action of CO is inhibited by hemoglobin (Hb), HO-1 no longer prevents EC apoptosis while these reagents do not affect the antiapoptotic action of bcl-2. Exposure of ECs to exogenous CO, under inhibition of HO-1 activity by SnPPIX, substitutes HO-1 in preventing EC apoptosis. The mechanism of action of HO-1/CO is dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) signaling transduction pathway. Expression of HO-1 or exposure of ECs to exogenous CO enhanced p38 MAPK activation by TNF-α. Specific inhibition of p38 MAPK activation by the pyridi...
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BACKGROUND: Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an ectoenzyme, which plays a role into several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of... more
BACKGROUND: Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an ectoenzyme, which plays a role into several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack of potency and specificity. Quinazolin-4-piperidin-4-methyl sulfamide (QPS) derivatives have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. METHOD: We have investigated the potency and selectivity of QPS derivatives in inhibiting NPP1 by enzymatic activities. The biological effect of QPS derivatives was documented on the mineralization of valve interstitial cell cultures, apoptosis assay, as well as quantitative polymerase chain reaction. RESULTS: We documented that QPS1 derivative is a potent (67.9 ±5.3 nM) and selective non-competitive inhibitor of human NPP1. Moreover, QPS1 also significantly inhibited the K121Q NPP1 gene variant (ki 51.9±9.8 nM), which is prevalent in the general population. QPS1 did not significantly alter the activity of other nucleotide metabolising enzymes expressed at the cell surface, namely NPP3, NTPDases (1-3), ecto-5’-nucleotidase and ALP. Importantly, QPS1 in the low micromolar range (≤10μM) prevented phosphate-induced mineralization of VICs and lowered the rise of osteogenic genes as expected for NPP1 inhibition. CONCLUSION: We provide evidence that QPS1 is a potent and selective non-competitive inhibitor of NPP1 that prevents pathologic mineralization in a cellular model.
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Purinergic signaling plays a major role in T cell activation leading to IL-2 production and proliferation. However, it is unclear whether purinergic signaling contributes to the differentiation and activation of effector T cells. In this... more
Purinergic signaling plays a major role in T cell activation leading to IL-2 production and proliferation. However, it is unclear whether purinergic signaling contributes to the differentiation and activation of effector T cells. In this study, we found that the purinergic receptor P2X4 was associated with human Th17 cells but not with Th1 cells. Inhibition of P2X4 receptor with the specific antagonist 5-BDBD and small interfering RNA inhibited the development of Th17 cells and the production of IL-17 by effector Th17 cells stimulated via the CD3/CD28 pathway. Our results showed that P2X4 was required for the expression of retinoic acid-related orphan receptor C, which is the master regulator of Th17 cells. In contrast, inhibition of P2X4 receptor had no effect on Th1 cells and on the production of IFN-γ and it did not affect the expression of the transcription factor T-bet (T-box transcription factor). Furthermore, inhibition of P2X4 receptor reduced the production of IL-17 but not of IFN-γ by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis. In contrast to P2X4, inhibition of P2X7 and P2Y11 receptors had no effects on Th17 and Th1 cell activation. Finally, treatment with the P2X4 receptor antagonist 5-BDBD reduced the severity of collagen-induced arthritis in mice by inhibiting Th17 cell expansion and activation. Our findings provide novel insights into the role of purinergic signaling in T cell activation and identify a critical role for the purinergic receptor P2X4 in Th17 activation and in autoimmune arthritis.
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BACKGROUND Hypertension is associated with a hypertrophic remodeling of big arteries, oxidative stress and inflammation. Extracellular nucleotides, released under stress condition, play a role in various physiopathological conditions... more
BACKGROUND Hypertension is associated with a hypertrophic remodeling of big arteries, oxidative stress and inflammation. Extracellular nucleotides, released under stress condition, play a role in various physiopathological conditions through P2 receptors activation. Hydrolysis of nucleotides is achieved by nucleoside triphosphate diphosphohydrolases, in particular NTPDase1 (CD39) which is highly expressed in the arterial wall. Together with ecto-5'nucleotidase (CD73), these ectoenzymes generate vasoprotective adenosine. The contribution of the purinergic signaling in cardiovascular diseases, hypertension in particular, remains to be established. Here, we investigate the potential benefit of extracellular nucleotidase in experimental hypertension by treating hypertensive mice with the potato ATPDase, Apyrase. METHODS Experimental hypertension was induced by Angiotensin II (AngII, subcutaneous 1mg/kg/day, osmotic pumps). Endogenous CD39 arterial expression and serum activity of hypertensive mice was evaluated by quantitative RT-PCR and by HPLC respectively (incubated with fluorescent etheno-ADP as a substrate). To replete mice with exogenous nucleotidase activity mice were treated or not with Apyrase (APY 90U/kg/day and 500U/kg ip every 3 days). Blood pressure was recorded daily by tail cuff plethys-mography until day 12 the day of the sacrifice. Then, media thickness of thoracic aorta was measured by histomorphometry, oxidative stress was evaluated with dihydroethidium staining and Immune cells infiltration was assessed by Immunofluorescence using pan immune cells marker (CD45) or macrophages marker (F4/80). Data were corroborated by quantitative RT-PCR. RESULTS We found a decrease in both CD39 arterial expression and serum activity in AngII-treated mice, suggesting a link of the endogenous ectonucleotidase CD39 to hypertension. In accordance with a protective role of nucleotidase activity, blood pressure increase and hypertrophic arterial remodeling were significantly reduced in AngII/APY-treated mice compared to AngII alone. Moreover, RT-qPCR and immunofluorescence experiments evidenced decreased perivascular immune cells infiltration and oxidative stress in AngII/APY-treated mice compared to AngII-treated mice. CONCLUSIONS Nucleotidase activities protect against vascular alterations associated to hypertension while the expression of CD39 decreases with the pathology. Nucleotidase may represent new therapeutic treatment for vascular diseases.
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We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the... more
We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of tr...
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Extracellular vesicles (EVs) are involved in cell-to-cell communication and modulation of numerous physiological and pathological processes. EVs are found in large quantities in milk and contain several inflammation- and... more
Extracellular vesicles (EVs) are involved in cell-to-cell communication and modulation of numerous physiological and pathological processes. EVs are found in large quantities in milk and contain several inflammation- and immunity-modulating proteins and microRNAs, through which they exert beneficial effects in several inflammatory disease models. Here, we investigated the effects of two EV subsets, concentrated from commercial cow’s milk, on a murine model of colitis induced with dextran sodium sulfate (DSS). P35K EVs, isolated by ultracentrifugation at 35,000 g, and P100K EVs, isolated at 100,000 g, were previously characterized and administered by gavage to healthy and DSS-treated mice. P35K EVs and, to a lesser extent, P100K EVs improved several outcomes associated to DSS-induced colitis, modulated the gut microbiota, restored intestinal impermeability and replenished mucin secretion. Also, P35K EVs modulated innate immunity, while P100K EVs decreased inflammation through the dow...
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Two isoforms of ATP diphosphohydrolase (ATPDase; EC 3.6.1.5 ) have been previously characterized, purified, and identified. This enzyme is an ectonucleotidase that catalyzes the sequential release of γ- and β-phosphate groups of... more
Two isoforms of ATP diphosphohydrolase (ATPDase; EC 3.6.1.5 ) have been previously characterized, purified, and identified. This enzyme is an ectonucleotidase that catalyzes the sequential release of γ- and β-phosphate groups of triphospho- and diphosphonucleosides. One of its putative roles is to modulate the extracellular concentrations of purines in different physiological systems. The purpose of this study was to define, identify, and localize these two isoforms of ATPDase in the pig digestive system. ATPDase activity was measured in pig stomach, duodenum, pancreas, and parotid gland. Enzyme assays, electrophoretograms, and Western blots with a polyclonal antibody that recognizes both isoforms demonstrate the presence of ATPDase in these organs. Immunolocalization showed intense reactions with gastric glands (parietal and chief cells), intestine (columnar epithelial cells), parotid gland, and pancreas. Smooth muscle cells all along the digestive tract were also highly reactive. ...
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An indiscriminate use of antibiotics in humans and animals has led to a widespread selection of antibiotic-resistant bacterial strains. A possible solution to counter this problem could be to develop alternatives that may boost the host... more
An indiscriminate use of antibiotics in humans and animals has led to a widespread selection of antibiotic-resistant bacterial strains. A possible solution to counter this problem could be to develop alternatives that may boost the host immunity, thus reducing in the quantity and frequency of antibiotic use. In this work, for the first time, citric acid and laccase were used as extracellular inducers of melanin production in yeast cells and human cell lines. It is proposed that the formulation of laccase and citric acid together could further promote melatonin stimulated melanocyte derived melanin production. Melanization test as a probe of immunity, described in this study, is an easy and a quicker test than the other immunity tests and is statistically significant. The results showed the synergistic effect of citric acid and laccase on melanin production by the yeast cells, with significant statistical differences compared to all other tested conditions (P: 0.0005-0.005). Laccase ...
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Many eukaryotic cells can felease ATP and other purines in their environment. ATP and its dephosphorylated derivatives could also be released as a result of cell permeabilization or cell breakage. These purines can bind to specific... more
Many eukaryotic cells can felease ATP and other purines in their environment. ATP and its dephosphorylated derivatives could also be released as a result of cell permeabilization or cell breakage. These purines can bind to specific receptors, called purinoceptors, and other proteins localized on the cell surface or on neighbouring cells1. In higher organisms that possess a circulatory system, ATP and other purines could move through the interstitial space and reach circulation. Hence, these purines may interact with cells localized very far from their site of origin. Work of the past few years has emphasized a diversity of purinoceptors that can bind ATP and its dephosphorylated derivatives and elicit a multiplicity of physiological actions. Indeed, purinoceptors have been found in the main physiological systems, namely: the circulatory system, the respiratory system, the digestive system, the nervous systems and the immune system2. In this context, the control of the purine concentration in vicinity of purinoceptors is of prime importance from the physiological viewpoint3.
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Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their... more
Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure-activity relationship, encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATP-α-thio-β,γ-CH2 (1), ATP-α-thio-β,γ-CCl2 (2), ATP-α-CH2-γ-thio (3), and 8-SH-ATP (4) and established their resistance to hydrolysis by NPP1,3 and NTPDase1,2,3,8 (<5% hydrolysis) (NTPDase = ectonucleoside triphosphate diphosphohydrolase). Analogues 1-3 at 100 μM inhibited thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis by NPP1 and NPP3 by >90% and 23-43%, respectively, and only slightly affected (0-40%) hydrolysis of ATP by NTPDase1,2,3,8. Analogue 3 is the most potent NPP1 inhibitor currently known, Ki = 20 nM and IC50 = 0.39 μM. Analogue 2a is a selective NPP1 inhibitor with Ki = 685 nM and IC50 = 0.57 μM. Analogues 1-3 were f...
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Circulatory homeostasis is usually maintained by quiescent endothelial cells that possess highly effective anticoagulant and platelet thromboregulatory mechanisms. Following injury, the vascular endothelium is considered to undergo a... more
Circulatory homeostasis is usually maintained by quiescent endothelial cells that possess highly effective anticoagulant and platelet thromboregulatory mechanisms. Following injury, the vascular endothelium is considered to undergo a process of activation where cells are exposed to oxidative stress, lose intrinsic antithrombotic properties and become procoagulant and facilitative for platelet aggregation. Endothelial cells express an ATPDase that hydrolyzes extracellular adenosine nucleotides and can inhibit stimulated human platelet aggregation in vitro. Loss of this ectoenzyme activity with reduced membrane protein expression occurs shortly following TNFα stimulation or perturbation of endothelial cells by reactive oxygen species in vitro. Comparable events follow reperfusion injury and xenograft rejection in vivo. Additionally, the administration of antioxidants and purified apyrases ameliorate rat renal reperfusion injury and discordant xenograft rejection, respectively.
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Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y2 mRNA receptor expression in the human colon adenocarcinoma cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues of patient suffering from... more
Inflammatory stresses associated with inflammatory bowel diseases up-regulate P2Y2 mRNA receptor expression in the human colon adenocarcinoma cell line Caco-2, the noncancerous IEC-6 cells and in colonic tissues of patient suffering from Crohn’s disease and ulcerative colitis. However, the transcriptional events regulating P2Y2 receptor (P2Y2R) expression are not known. We have identified a putative transcription start site in the P2Y2R gene and demonstrated acetylation of Lys14 on histone H3 and Lys8 on histone H4, thus suggesting that the chromatin associated with the P2Y2 promoter is accessible to transcription factors. We also showed that the transcription factor NF-κB p65 regulates P2Y2R transcription under both proinflammatory and basal conditions. A NF-κB-responsive element was identified at −181 to −172 bp in the promoter region of P2Y2. Hence, activation of P2Y2R by ATP and UTP stimulated cyclooxygenase-2 expression and PGE2 secretion by intestinal epithelial cells. These f...
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ABSTRACT Extracellular nucleotides promote vascular constriction through cell membrane P2 receptors. This effect involves neurogenic activation of vascular smooth muscle cell P2X1 (ATP) and some pyrimidine-sensitive (UDP, UTP) P2Y... more
ABSTRACT Extracellular nucleotides promote vascular constriction through cell membrane P2 receptors. This effect involves neurogenic activation of vascular smooth muscle cell P2X1 (ATP) and some pyrimidine-sensitive (UDP, UTP) P2Y receptors. We used knockout mouse models to unravel the role of extracellular nucleotides in myogenic tone of resistance arteries. The contractile effects of exogenous UDP and UTP is abrogated in P2RY6-/- arteries suggesting that P2Y6 receptor fully underlies vascular contraction to uracyl nucleotides. Moreover, the deletion of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1 or CD39) the dominant ectoenzyme hydrolysing nucleotides at the smooth muscle surface (Entpd1-/- mice) unmasks potent constrictor effect of UDP and UTP in conductance (thoracic aorta) and resistance (mesenteric) arteries. Mirroring these observations, myogenic tone was diminished in P2RY6-/- while it was exaggerated in Entpd1-/- mesenteric arteries. This suggests some autocrine release of extracellular nucleotides participates in resistance arteries autoregulation trough P2Y6 receptor activation. Indeed, extracellular nucleotides release is well known to occur in response to cell strain (stretch). Involvement of Panx1 in this release is likely and was corroborated by pharmacological approach as well as dye uptake experiments. Finally, we propose that this signalling by extracellular nucleotides may contribute to the regulation of blood pressure in pathological conditions since P2RY6-/- mice are partially resistant to experimental hypertension.
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Research Interests: Biochemistry, Chemistry, Organic Chemistry, Enzyme Inhibitors, Cytotoxicity, and 13 moreMedicine, Macromolecular X-Ray Crystallography, Humans, Animals, Enzyme, Rats, Cell Proliferation, Structure activity Relationship, Recombinant Proteins, Antineoplastic Agents, Molecular Structure, Vincristine, and Pharmacology and pharmaceutical sciences
ABSTRACT Extracellular nucleotide stimulation of purinergic/pyrimidinergic type-2 (P2) receptors are components of platelet, endothelial cell (EC), and leukocyte activation that culminate in vascular thrombosis and inflammation in vivo.... more
ABSTRACT Extracellular nucleotide stimulation of purinergic/pyrimidinergic type-2 (P2) receptors are components of platelet, endothelial cell (EC), and leukocyte activation that culminate in vascular thrombosis and inflammation in vivo. CD39, the prototype nucleoside triphosphate diphosphohydrolase (or NTPDase-1), is highly expressed on quiescent endothelium, monocytes, and activated lymphocytes and therefore could influence these pathways. The potential of NTPDase-1 to regulate P2-receptor function in the vasculature has been established by our generation of cd39-null mice. These mice exhibit a prothrombotic vascular phenotype ascribed to overexpression of tissue factor by endothelial cells following aberrant P2- (and potentially adenosine 2a/3) receptor activation. Mutant mice also show perturbations in hemostasis, secondary to platelet P2Y1-receptor desensitization. In addition, administration of soluble NTPDase and/or induction of CD39 overexpression by adenoviral vectors consistently result in amelioration of vascular injury in several animal models tested. CD39 is also the major NTPDase expressed by monocyte-macrophages (Mo). Upregulation of tissue factor expression by Mo in vitro and alterations in splenic populations in vivo have been observed in cd39-null mice. Paradoxical inhibition of integrin-mediated adhesion and transendothelial migration of cd39-null Mo are also related to aberrant P2-receptor activation and have also been observed in vitro and in vivo. Overexpression of CD39 following infection with recombinant adenoviral vectors also blocks LPS-induced ATP secretion and inhibits IL-1 release in vitro. These studies confirm a role for CD39 in the differential regulation of P2-receptor activity and function in platelets, vascular, and immune cells. Drug Dev. Res. 53:193–207, 2001. © 2001 Wiley-Liss, Inc.
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Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an ectoenzyme, which plays a role in several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack... more
Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an ectoenzyme, which plays a role in several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack potency and specificity. Quinazolin-4-piperidine sulfamides (QPS) have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. In the present series of experiments, we have evaluated the efficacy of two derivatives, QPS1-2, in inhibiting human NPP1, and we have evaluated the effect of the most potent derivative (QPS1) on other ectonucleotidases as well as on the ability of this compound to prevent phosphate-induced mineralization of human primary aortic valve interstitial cells (VICs). We documented that QPS1 derivative is a potent (Ki 59.3±5.4 nM) and selective non-competitive inhibitor of human NPP1. Moreover, QPS1 also significa...
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Research Interests: Electron Microscopy, Biology, Cell Biology, Biological Chemistry, Medicine, and 15 moreBiological Sciences, Humans, Mutagenesis, Animals, Biological, CHEMICAL SCIENCES, Caveolae, Palmitoylation, Membrane Protein, Cytoplasm, Plasma Membrane, Palmitic Acid, Nucleotides, Cell Membrane, and Medical and Health Sciences
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Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults and the current treatments only have a modest effect on patient survival. Recent studies show that bozepinib (BZP), a purine derivative, has potential... more
Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults and the current treatments only have a modest effect on patient survival. Recent studies show that bozepinib (BZP), a purine derivative, has potential applications in cancer treatment. The aim of this study was to evaluate the effect of BZP against GBM cells, specially concerning the purinergic system. Thus, GBM cells (C6 and U138 cell lines) were treated with BZP and cell viability, cell cycle, and annexin/PI assays, and active caspase-3 measurements were carried out. Besides, the effect of BZP over the purinergic system was also evaluated in silico and in vitro. Finally, we evaluate the action of BZP against important markers related to cancer progression, such as AKT, NF-κB, and CD133. We demonstrate here that BZP reduces GBM cell viability (IC50 = 5.7 ± 0.3 µM and 12.7 ± 1.5 µM, in C6 and U138 cells, respectively), inducing cell death through caspase-dependent apoptosis, autophagosome formation, activation of NF-κB, without any change in cell cycle progression or on the Akt pathway. Also, BZP modulates the purinergic system, inducing an increase in CD39 enzyme expression and activity, while inhibiting CD73 activity and adenosine formation, without altering CD73 enzyme expression. Curiously, one cycle of treatment resulted in enrichment of GBM cells expressing NF-κB and CD133+, suggesting resistant cells selection. However, after another treatment round, the resistant cells were eliminated. Altogether, BZP presented in vitro anti-glioma activity, encouraging further in vivo studies in order to better understand its mechanism of action.